Numerous reports of multi-drug resistant bacterial strains have appeared in recent years, with several strains posing the threat of becoming immune against all commercially available antibiotics. In order to prevent potential epidemic outbreaks of infectious diseases, a renewed focus on antibiotic research is highly desired, including the search for new natural products with alternative cellular targets, the investigation of the mechanisms of cytotoxicity and resistance, and the understanding of their biosynthetic pathways. Relatively unexplored with respect to biosynthetic pathways are phosphonate antibiotics, despite their potential use in antibacterial, antiviral, and antiparasitic therapies. Moreover, they are used extensively in agriculture as herbicides and pesticides. This project aims to biochemically characterize the biosynthetic pathways of several of these compounds including bialaphos (phosphinothricin tripeptide) and A53868 using the gene sequence information available from collaborators within this program project. Proteins will be expressed in E. coli or S. lividans and purified and their putative substrates will be chemically synthesized. In addition to elucidating the biosynthetic pathway, the mechanism of a select group of unusual enzymes will also be investigated. This project will also augment the natural phosphonates discovered by genetic studies with synthetic biased libraries that will focus on analogs of natural occurring phosphonates. The initial libraries will focus on K-26 and A53868.